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Dedifferentiation Alters Chondrocyte Nuclear Mechanics During in Vitro

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Dedifferentiation Alters Chondrocyte Nuclear Mechanics During in Vitro bioRxiv preprint doi: https://doi.org/10.1101/2021.04.26.441500; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dedifferentiation alters chondrocyte nuclear mechanics during in vitro culture and expansion Authors Soham Ghosh 1, 2, *, Adrienne K. Scott 3, Benjamin Seelbinder 3, Jeanne E. Barthold 3, Brittany M St. Martin 3, Samantha Kaonis 2, Stephanie E. Schneider 3, Jonathan T. Henderson 4, Corey P. Neu 3 Affiliations 1 Department of Mechanical Engineering, Colorado State University, Fort Collins, CO 2 School of Biomedical Engineering, Colorado State University, Fort Collins, CO 3Department of Mechanical Engineering, University of Colorado Boulder, Boulder, CO 4 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN * Correspondence: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.04.26.441500; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT Dedifferentiation of chondrocytes during in vitro passaging before implantation, and post implantation in vivo, is a critical limitation in cartilage tissue engineering. Several biophysical features define the dedifferentiated state including a flattened cell morphology and increased stress fiber formation. However, how dedifferentiation influences nuclear mechanics, and the possible long-term implications of this state, are unknown. In this study, we investigated how chondrocyte dedifferentiation affects the mechanics of the chromatin architecture inside the cell nucleus and the gene expression of the structural proteins located at the nuclear envelope. Through an experimental model of cell stretching and a detailed spatial intranuclear strain quantification, we identified that strain is amplified and distribution of strain within the chromatin is altered under tensile loading in the dedifferentiated state. Further, using a confocal microscopy image-based finite element model and simulation of cell stretching, we found that the cell shape is the primary determinant of the strain amplification inside the chondrocyte nucleus in the dedifferentiated state. Additionally, we found that nuclear envelope proteins have lower gene expression in the dedifferentiated state suggesting a weaker nuclear envelope which can further intensify the intranuclear strain amplification. Our results indicate that dedifferentiation and altered nuclear strain could promote gene expression changes at the nuclear envelope, thus promoting further deviation from chondrocyte phenotype. This study highlights the role of cell shape on nuclear mechanics and lays the groundwork to design biophysical strategies for the maintenance and enhancement of the chondrocyte phenotype during expansion with a goal of successful cartilage tissue engineering. bioRxiv preprint doi: https://doi.org/10.1101/2021.04.26.441500; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. SIGNIFICANCE Chondrocytes dedifferentiate into a fibroblast-like phenotype in a non-native biophysical environment. Using high resolution microscopy, intranuclear strain analysis, finite element method based computational modeling, and molecular biology techniques, we investigated how mechanical force causes abnormal intranuclear strain distribution in chondrocytes during the dedifferentiation process. Overall, our results suggest that the altered cell geometry aided by an altered or weakened nuclear envelope structure are responsible for abnormal intranuclear strain during chondrocyte dedifferentiation that can further deviate chondrocytes to a more dedifferentiated state. bioRxiv preprint doi: https://doi.org/10.1101/2021.04.26.441500; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. INTRODUCTION Cartilage tissue engineering has received specific attention in the attempt to treat osteoarthritis, a debilitating disease affecting a large worldwide population (1). Pharmaceutical therapies to treat this degenerative disease are limited, and current disease management protocol relies on lifestyle changes and the use of drugs to ameliorate pain or to reduce inflammation. Tissue engineering and regenerative medicine are promising approaches with the goal to rebuild the damaged articular cartilage to restore the functions of the knee joint (2). Autologous Chondrocyte Implantation (ACI) is a promising approach for cartilage tissue engineering, where chondrocytes extracted from the patient are expanded in vitro and then implanted back in the knee joint to regenerate functional articular cartilage (2–4). However, there are two critical challenges that limit the potential widespread application of this technology. First, from clinical follow up studies, it is now apparent that the success of the ACI treatment is limited because of post-operative fibrosis that leads to inferior biomechanical and functional response of the knee joint (5–9). The post-operative fibrosis is probably triggered by the dedifferentiation of the chondrocytes to fibroblast-like phenotype after implantation because the chondrocytes experience a mechanically fibrous and stiffer tissue microenvironment different than the native healthy cartilage environment, not conducive to the maintenance of the chondrocyte phenotype. The second critical challenge of ACI involves the expansion of the chondrocytes in vitro. During the expansion process and passaging, the chondrocytes dedifferentiate into fibroblast-like cells while cultured on a two- dimensional (2D) monolayer culture. Consequently, the scalability of the ACI technology is limited because growing enough cells for a large defect requires extensive time, which can further dedifferentiate chondrocytes in 2D culture. bioRxiv preprint doi: https://doi.org/10.1101/2021.04.26.441500; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Changes in mechanical loading of chondrocytes might lead to chondrocyte dedifferentiation in vivo and in vitro. Static and dynamic loading on the chondrocyte and its complex interaction with the pericellular matrix (PCM) and extracellular matrix (ECM) probably plays a critical mechanobiological role (10–13) in the dedifferentiation process (14). Mechanical stretching on chondrocytes in the native cartilage environment is shown to induce several pathophysiological responses mediated by several mechanobiological pathways (15– 17). In vitro, the monolayer culture of chondrocytes on a dish imparts a passive two- dimensional mechanical stretch on the cell, aided by the flat cell shape and contractile forces imparted by F-actin stress fibers. Dedifferentiated chondrocytes in monolayer culture have a higher cell stiffness mediated by a cell membrane-F actin adhesion mechanism (18), indicating higher contractile forces within the cell. Interestingly, several studies indicate that if the cells are instead encapsulated in a three-dimensional (3D) environment, the chondrocyte phenotype prevails. Even the late passage chondroblasts (a term used to define a cell showing the phenotype of both chondrocyte and fibroblast) redifferentiate into chondrocytes (19, 20) when cultured in 3D, suggesting that releasing cell contraction experienced in 2D ameliorates the dedifferentiation. The mechanical loading not only affects the chondrocyte cytoskeletal mechanics, but also it reaches and affects the cell nucleus as well (21–23) which might have unknown implications in the chondrocyte dedifferentiation process. The mechanics of the cell nucleus and its biological implication, termed as nuclear mechanobiology has recently been shown to be important in numerous biological contexts - in diseases (24, 25), gene expression (26), differentiation (27, 28), developmental biology (29) and tissue homeostasis (30). The study of nuclear mechanobiology investigates how the mechanical microenvironment (i.e., stretch, compression and shear) affects the spatiotemporal function of the nuclear envelope, the nucleoskeleton, the chromatin architecture, and hence the gene regulation. A recent study bioRxiv preprint doi: https://doi.org/10.1101/2021.04.26.441500; this version posted April 27, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. elucidated that the loss of extracellular matrix stiffness and the lack of three-dimensional chondrocyte matrix decreases the chondrocyte cell and nuclear
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